LOCUS       sequence-471878-        6486 bp    DNA     circular SYN 14-JUL-2025
DEFINITION  Express hVPS4B-HaloTag(R) Fusion Protein in Mammalian Cells under a 
            CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6486)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 6486)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: hVPS4B-HaloTag Vector
FEATURES             Location/Qualifiers
     source          1..6486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    3..84
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     enhancer        317..620
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        621..824
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     775..795
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     intron          960..1092
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     1075..1094
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     primer_bind     1136..1155
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1136..1154
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1991..2002
                     /codon_start=1
                     /product="WELQut protease recognition and cleavage site"
                     /label=WELQut site
                     /translation="WELQ"
     CDS             2521..2541
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="EDLYFQS"
     CDS             2557..3441
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="EIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSSYV
                     WRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVIHD
                     WGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLIIDQ
                     NVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALVEE
                     YMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDLIG
                     SEIARWLSTLEISG"
     polyA_signal    3528..3649
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3539..3558)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     protein_bind    complement(3764..3797)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        3807..3854
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3886..4680
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3940..3959)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4550..4569
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     rep_origin      complement(4838..5426)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4918..4937)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    5552..5600
                     /note="synthetic polyadenylation signal"
     CDS             complement(5624..6022)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      6019..6028
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6053..6410)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      6067..6202
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        6219..6410
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     6367..6387
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
ORIGIN
        1 cggcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc
       61 attataagct gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt
      121 cagggggagg tgtgggaggt tttttaaagc aagtaaaacc tctacaaatg tggtatggct
      181 gattatgatc cgcggccgca atacgtcgac gttatcagct gacttcgtac gagagcctag
      241 gattatggcg cgccactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
      301 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      361 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      421 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      481 aagtgtatca tatgccaagt ccgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      541 ggcattatgc ccagtacatg accttacggg actttcctac ttggcagtac atctacgtat
      601 tagtcatcgc tattaccatg gtgatgcggt tttggcagta caccaatggg cgtggatagc
      661 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      721 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaataa ccccgccccg ttgacgcaaa
      781 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc
      841 agatcactag aagctttatt gcggtagttt atcacagtta aattgctaac gcagtcagtg
      901 cttctgacac aacagtctcg aacttaagct gcagaagttg gtcgtgaggc actgggcagg
      961 taagtatcaa ggttacaaga caggtttaag gagaccaata gaaactgggc ttgtcgagac
     1021 agagaagact cttgcgtttc tgataggcac ctattggtct tactgacatc cactttgcct
     1081 ttctctccac aggtgtccac tcccagttca attacagctc ttaaggctag agtattaata
     1141 cgactcacta tagggctagc gatcgccatg tcatccactt cgcccaacct ccagaaagcg
     1201 atagatctgg ctagcaaagc agcgcaagaa gacaaggctg ggaactacga agaagccctt
     1261 cagctctatc agcatgctgt gcagtatttt cttcatgtcg ttaaatatga agcacagggt
     1321 gataaagcca agcaaagtat cagggcaaag tgtacagaat atcttgatag agcagaaaaa
     1381 ctaaaggagt acctgaaaaa taaagagaaa aaagcacaga agccagtgaa agaaggacag
     1441 ccgagtccag cagatgagaa ggggaatgac agtgatgggg aaggagaatc tgatgatcct
     1501 gaaaaaaaga aactacagaa tcaacttcaa ggtgccattg ttatagaacg accaaatgtg
     1561 aaatggagtg acgttgctgg acttgaagga gccaaagaag cactgaaaga ggctgtgata
     1621 ctgcctatta aatttcctca tctttttaca ggcaagagaa caccttggag gggaatccta
     1681 ttatttgggc cgcctggaac aggaaagtcc tacttagcca aagctgtagc aacagaagcc
     1741 aacaactcaa catttttttc aatatcttcc tctgatcttg tttctaagtg gctaggtgaa
     1801 agtgaaaaac tggttaagaa tttattccaa cttgccagag agaacaagcc ctccattatc
     1861 ttcattgatg aaattgattc tctctgtggt tcaagaagtg aaaatgaaag tgaagccgca
     1921 cgtagaatta agacggagtt cctagtgcaa atgcaagggg ttggtgtaga caatgatgga
     1981 attttggttc tgggagctac aaatataccc tgggttctgg attctgccat taggcgaaga
     2041 tttgagaaac gaatttatat tcccttgccg gaaccccatg cccgagcagc aatgtttaaa
     2101 ctgcacctag ggaccactca gaacagtctc acggaagcag actttcggga acttgggagg
     2161 aaaacagatg gttattcagg ggcagatata agtatcattg tacgtgatgc ccttatgcag
     2221 cctgttagga aagtacagtc agctactcat tttaaaaagg ttcgcggacc ttcccgagct
     2281 gatcctaacc atcttgtaga tgatctgcta acaccttgct ctccaggtga ccctggtgcc
     2341 attgaaatga catggatgga tgtccctgga gataaacttt tggagccagt tgtttccatg
     2401 tcggatatgt tgcggtcact atctaacaca aaacctacag tcaatgaaca tgacttgttg
     2461 aaattaaaga agtttacaga agattttggt caagaaggcg tttctctcga gccaaccact
     2521 gaggatctgt actttcagag cgataacgat ggatccgaaa tcggtactgg ctttccattc
     2581 gacccccatt atgtggaagt cctgggcgag cgcatgcact acgtcgatgt tggtccgcgc
     2641 gatggcaccc ctgtgctgtt cctgcacggt aacccgacct cctcctacgt gtggcgcaac
     2701 atcatcccgc atgttgcacc gacccatcgc tgcattgctc cagacctgat cggtatgggc
     2761 aaatccgaca aaccagacct gggttatttc ttcgacgacc acgtccgctt catggatgcc
     2821 ttcatcgaag ccctgggtct ggaagaggtc gtcctggtca ttcacgactg gggctccgct
     2881 ctgggtttcc actgggccaa gcgcaatcca gagcgcgtca aaggtattgc atttatggag
     2941 ttcatccgcc ctatcccgac ctgggacgaa tggccagaat ttgcccgcga gaccttccag
     3001 gccttccgca ccaccgacgt cggccgcaag ctgatcatcg atcagaacgt ttttatcgag
     3061 ggtacgctgc cgatgggtgt cgtccgcccg ctgactgaag tcgagatgga ccattaccgc
     3121 gagccgttcc tgaatcctgt tgaccgcgag ccactgtggc gcttcccaaa cgagctgcca
     3181 atcgccggtg agccagcgaa catcgtcgcg ctggtcgaag aatacatgga ctggctgcac
     3241 cagtcccctg tcccgaagct gctgttctgg ggcaccccag gcgttctgat cccaccggcc
     3301 gaagccgctc gcctggccaa aagcctgcct aactgcaagg ctgtggacat cggcccgggt
     3361 ctgaatctgc tgcaagaaga caacccggac ctgatcggca gcgagatcgc gcgctggctg
     3421 tctactctgg agatttccgg ttaatagaat tctagagtcg acctgcaggc atgcaagctg
     3481 atttggctgc tgccaccgct gagcaaggcc gcttcgagca gacatgataa gatacattga
     3541 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     3601 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     3661 ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt taagcaagta
     3721 aaacctctac aaatgtggta aaatcttaat taagaattct aatataactt cgtatagcat
     3781 acattatacg aagttatgga tccactgttg acaattaatc atcggcatag tatatcggca
     3841 tagtataata cgacaaggtg aggaactaaa cccaggaggc agatcatgat tgaacaagat
     3901 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
     3961 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
     4021 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg
     4081 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
     4141 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
     4201 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
     4261 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
     4321 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
     4381 gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc
     4441 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
     4501 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     4561 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     4621 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     4681 ttcgaaatga ccgaccaagc gacgcccaac cggtatcagc tcactcaaag gcggtaatac
     4741 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     4801 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     4861 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     4921 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     4981 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5041 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5101 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5161 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5221 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5281 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5341 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     5401 ttacgcgcag aaaaaaagga tttcaagaag atcctttgat cttttctacg gggtctgacg
     5461 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     5521 tcacctagat ccttttatag tccggaatcg tcacacaaaa aaccaacaca cagatgtaat
     5581 gaaaataaag atattttatt gcggccatcg tgatggctag agtttagccc tcccacacat
     5641 aaccagaggg cagcaattca cgaatcccaa ctgccgtcgg ctgtccatca ctgtccttca
     5701 ctatggcttt gatcccagga tgcagatcga gaagcacctg tcggcaccgt ccgcaggggc
     5761 tcaagatgcc cctgttctca tttccgatcg cgacgataca agtcaggttg ccagctgccg
     5821 cagcagcagc agtgcccagc accacgagtt ctgcacaagg tcccccagta aaatgatata
     5881 cattgacacc agtgaagatg cggccgtcgc tagagagagc tgcgctggcg acgctgtagt
     5941 cttcagagat ggggatgctg ttgattgtag ccgttgctct ttcaatgagg gtggattctt
     6001 cttgagacaa aggcttggcc atggtggcgc tagtgtcaga agaatcgagc tttttgcaaa
     6061 agcctaggcc tccaaaaaag cctcctcact acttctggaa tagctcagag gccgaggcgg
     6121 cctcggcctc tgcataaata aaaaaaatta gtcagccatg gggcggagaa tgggcggaac
     6181 tgggcggagt taggggcggg atgggcggag ttaggggcgg gactatggtt gctgactaat
     6241 tgagatgcat gctttgcata cttctgcctg ctggggagcc tggggacttt ccacacctgg
     6301 ttgctgacta attgagatgc atgctttgca tacttctgcc tgctggggag cctggggact
     6361 ttccacaccc taactgacac acattccaca gctggttctt tccgcctcag aaggtaccta
     6421 accaagttcc tctttcagag gttatttcag gccatggtgc tgcgcaagac gcgttaatta
     6481 agacgt
//
